Anti-CD6 monoclonal antibodies and their uses

ABSTRACT

Monoclonal antibodies that recognize the CD6 antigen, pharmaceutical compositions that recognizes and that are able to achieve a clinical and histological effectivity in patients with different clinical types of Psoriasis.

FIELD OF THE INVENTION

The present invention is related to the field of immunology andparticularly with obtaining a pharmaceutical composition that contains amonoclonal antibody that recognizes the CD6 leukocyte differentiationantigen.

DESCRIPTION OF THE PRIOR ART

Monoclonal antibodies (mAbs) have permitted the characterization ofmolecules of physiological importance expressed on the cell surface.Defined in the cells of the Immune System the “Leukocyte DifferentiationClusters” or antigens (CD) (scholossman, S. F. et al. (1994) Immunol.Today 15 (3);98). The definition of the role of the CD′s in thedifferentiation and maturation of the lymphoid cells during theirontogenic development, in the mechanisms of cellular recognition andadhesion and in the mechanisms of activation and proliferation duringthe immune response have conducted to the use of their respective mAbsin diagnosis and immunotherapy, with promising results (Dantal, J. etal. (1991) Curr. Opin. Immunol. 3:740).

The murine mAbs directed against molecules expressed on the cellmembrane of human T lymphocytes have contributed to improve thediagnosis of clinical entities in which there is dysfunction of thesecells. Moreover they have been used to explore new therapeutic approachswith the purpose of modulating their functional activity as in the casesof Transplant Rejection, Graft Versus Host Disease (GvHD) and AutoimmuneDiseases (Waldman, T. A. (1991) Science 252:1657; Waldman, T. A. (1992)Annu. Rev. Immunol. 10: 675).

The CD6 is a not well characterized molecule. It is known that it is aglycoprotein existing in two molecular forms maintained in dynamicequilibrium and differing only in the grade of phosphorylation. In theresting T lymphocytes it is a phosphorylated molecule of 105 kDa, whilein activated cells it is a hyperphosphorylated form of 130 kDa(Cardenas, L. et al. (1990) J. Immunol. 145:1450; Swack, J. A. et al.(1991) J. Biol. Chem. 266: 7137), member of a family of membranereceptors and secretion proteins with a characteristic structure(Kodama, T. et al. (1990) Nature 343: 531; Aruffo, A. et al. (1991) J.Exp. Med. 174: 949). It is expressed on the surface of mature humanthymocytes, in T lymphocytes of peripheral blood, constituting themajority of the CD3+ cell population, in a subtype of B lymphocytes andin the neurons of the brain cortex. In the T lymphocytes of peripheralblood it participates in the mechanisms of cellular activation(Reinhertz, E. L. et al. (1982) Cell 30: 735; Kamoun, M. et al (1981) J.Immunol. 127: 987; Mayer, B. et al. (1990) J. Neuroimmunol. 29: 193;Rasmussen, R. A. et al. (1994) J. Immunol. 152: 527).

The role of the CD6 molecule in the T cell ontogenesis as well as itspossible role in the physiopathology of diseases of different etiologyare unknown.

Recently a CD6 ligand was identified and characterized having anextensive cellular distribution in normal tissues such as thymus,spleen, lymph nodes, and skin (Dhavalkumar, D. P. et al. (1995) J. Exp.Med. 181:1563). This molecule, denominated ALCAM (ActivatedLeukocyte-Cell Adhesion Molecule due to its expression in activated Tand B lymphocytes as well as monocytes, is a 100 kDa molecular weighttype I membrane glycoprotein with five extracellular domains similar tothose of the immunoglobulins. It can present different activation levelsdepending on divalent cations and can mediated heterophylic andhomophylic interactions (Bowen, M. A. et al. (1995) J. Exp Med. 181:2213).

Different anti CD6 mAbs have been used in clinical research for theprevention of the rejection crisis in organ transplantation (Kirkman, R.L. et al. (1983) Transplantation 36: 620) and to deplete bone marrowtransplants from lymphocytes for preventing Graft versus Host Disease(GVHD) (Soiffer, R. J. et al. (1992) J. Clin. Oncol. 10:1191). The iort1 mAb is in a Phase II Clinical Trial for the treatment of CutaneousT-Cell Lymphomas (Garcia, C. A. et al. (1990) Biotecnologia Aplicada7(2):176; Faxas, M. E. et al (1993) Biotecnologia Aplicada 10(1): 20).

The ior t1 monoclonal antibody of IgG2a isotype was classified as antiCD6 in the IV international Workshop of Leucocyte DifferentiationAntigens, Vienna (1989). This mAb defines an epitope different from theones recognized by other anti CD6 mAbs. The epitope has a stableconformation and is insensitive to reduction agents, being possiblylocated in the primary structure of the CD6 molecule (Osorio, L. M. etal. (1994) Cell Immunol. 154:123).

This monoclonal antibody has a lower recognition than other CD6 mAbs inperipheral mononuclear cells of healthy donors. The recognition patternof ior t1 mAb in human cell culture lines of T lymphocytes origin is 47%in Jurkat cells, 23% in Molt-4 cells and no recognition of CCRF-CEMcells; of B lymphocytes origin is 9% in Raji, of erythroblastoid originis 12% in K-562 and of myelomonocytic origin is 9% in U-937. It alsorecognizes peripheral mononuclear cells of Chronic B LymphocyticLeukemia (89+/−4%) (Garcia C. A. et al. 1992) Biotecnologia Aplicada9(1):70) and lymphocytes of cutaneous lesions in patients with CutaneousT-Cell Lymphomas (Rodriguez, T. et al. (1985) Interferon y Biotec. 2(1):41).

The ior t1 mAb does not inhibit the in vitro antigen specific cellularcytotoxicity (Faxas, M. E. et al. (1993) Biotecnologia Aplicada10(1):47). It is capable of activating in vitro peripheral blood Tlymphocytes of healthy donors. At suboptimum concentrations of OKT3(anti CD3) the cross-linking with ior t1 induces higher responses thanthose achieved with other anti CD6 mAbs (Osorio, L. M. et al. (1994)Cell Immunol. 154:123).

Psoriasis is a disease whose physiopathology has not been defined(Hunziker, T. et al. (1993) Ther. Umsch. 50(2);110; Elder, J. T. et al.(1994) J. Invest. Dermatol. 102(6): 24S). It is characterized bypresenting an inflammatory infiltrate in the target organ withpredominance of activated T lymphocytes of CD4+ and CD8+ phenotypes(Chang, J. C. C. et al. (1994) Proc. Natl. Acad. Sci. USA 91:9282), aswell as strong olygoclonality of the T-Cell receptors, the cells seem topresent a marked tendency to migrate to the skin (homing) (Baker, J. N.W. N. et al (1992) Br. J. Dermatol. 127; 205; Menssen, A. et al. (1995)J. Immunol. 155:4078; Valdimarsson, H. et al. (1995) Immunol. Today16(3):145).

The spontaneous remission of Psoriasis can be predicted if the number ofT cells in the skin decrease. Thus, it is suggested they play animportant role in the perpetuation of the disease by releasing solublemediators of the immune response capable of inducing the proliferationof keratinocytes, responsible of clinical manifestations of the disease.

These considerations are supported by facts such as the cure of thedisease following allogeneic bone marrow transplantation, the possibleHLA association, the improvement, with steroids and specially withimmunosupressocs like cyclosporine and the clinical improvement,although reversible, with anti T cells therapeutic monoclonal artibodies(Griffiths, C. E. M. et al. (1992) Springer Semin Immunopathol. 13:441;Nanney, L. B. et al. (1986) J. Invest. Dermatol. 86(3): 260; Ficassia,D. D. et al. (1987) J. Am. Acad. Dermatol 17)3: 408; Schopf, R. E. etal. (1986) Arch. Dermatol. Res. 279(2 ): 89; van de Kerkhof, P. C. etal. (1997) Dermatologica 174(5): 224).

The success o immunotherapy with monoclonal antibodies depends on theselection of the target molecule, which should participate in importantcellular functions or in the selection of the mAb (Dantal, J. et al.(1991) urr. Opin. Immunol. 3:740). The mAbs evaluated in Psoriasisdirected against CD3 (Weinshenker, B. G. at al. (1989) J. Am. Acad.Dermatol. 20:1132) and against CD4 (Poizot-Martin, I. et al. (1991)Lancet 337:1477; Prinz J. et al. (1991) Lancet 338: 320; Nicolas, J. F.et al. (1991) Lancet 338: 321) have produced clinical improvement in thepatients after multiple high dose endovenous applications, with partialremissions of short duration and early relapse of the symptoms and signsof the disease in all cases.

Therapeutical application of murine mAbs in multiple doses is associatewith some secundary effects and limited clinical utility in patients,these are related with xenogenicity of murine proteins that induce anHuman Anti-Mouse Antibodies response (HAMA): humanized antibodiesgenerated by protein engineering have reduced immunogenicity, improvedpharmacokinetics and clinical advantage (Winter, G. et al. (1993)Trends-Pharmacol-Sci. 14(5): 139).

Up to now no previous study describing the expression of the CD6molecule in the T lymphocytes of the inflammatory infiltrate of the skinin Psoriasis nor the possible association of this molecule with thedevelopment of the disease has been reported. Additionally thetherapeutic use of an anti CD6 n in this disease has not been previouslyevaluated.

The novelty of the present invention consists in providing topical andsystemic pharmaceutical compositions containing an anti CD6 monoclonalantibody and the obtention of an humanized monoclonal antibody anti CD6for its application in patients with Psoriasis Vulgar, using differentadministration routes and in different clinical forms of the disease.

DETAILED DESCRIPTION OF THE INVENTION OBTAINMENT OF THE MONOCLONALANTIBODY

Purification of the Murine Monoclonal Antibody.

The anti CD6 monoclonal antibody (mAb) can be purified from ascitisfluid by Protein A Sepharose, diluted with an equal volume of glycinebuffer 1.5 M, NaCl 3 M pH 0.9, equilibrating the matrix with the samebuffer. Applying equal volumes of ascitis and buffer elution should beperformed at flow speed of 50 mL/h. Wash the column overnight until thezero base line, with the same application buffer.

Afterwards the column is washed with citric acid buffer 0.1 M pH 6 toeliminate the IgG1 immunoglobulins, until the base line reaches zero, ata flow speed of 50 mL/h (between 2 and 5 column volumes, approximately 2hours). Buffer citric acid 0.1 M, pH5 is applied to elute the IgG2aimmunoglobulins (ior t1).

Humanization of the Murine Monoclonal Antibody

Using genetic engineering methods variants of the anti CD6 murine mAbscan be constructed, such as chimaeric and humanized antibodies, from thevariable regions of the light and heavy chains of the murine antibody(Takashi, N. et al. (1982) Cell 29:718; Hieter, P. A. et al. (1980) Cell29:718).

Description of Method of Humanization of the Murine Monoclonal Antibodyior t1.

A subclone ior t1A was obtained from the parental murine ior t1secreting hybridoma cells which recognize the same epitope on the CD6molecule.

ior t1A was modified in order to decrease its immunogenicity with aprocedure (patent # 0699755 E.P. Bul.) Which simultaneously reducesimmunogenicity of the rodent monoclonal antibody while preserving itsligand binding properties in their entirety. Since the antigenicity ofan immunoglobulin is dependent on the presence of T-cell antiganicpeptides onto their sequence, the immunogenicity of a xenogenic orallogenic antibody could be reduced by replacing the residues includedonto the T-cell antigenic sequences which differ from those usuallyfound in another mammalian species antibodies. Of course, thereplacement of residues do not include those involved in to thecanonical structures or in the Vernier zone. This judicious replacementof residues have no effect on the structural determinants or on theinterdomain contacts, thus, ligand binding properties should beunaffected as a consequence of alterations which are limited to thevariable region framework residues.

Analysis of Homology of Variable Regions:

The present procedure makes use of the available sequence data for humanantibody variable domains compiled by Kabat et al., “Sequences ofproteins of Immunological Interest” Fifth edition., Bethesda, Md.;National Inst. of Health, 1994.

In the first step the variable domains of the murine ior t1 heavy orlight chain are compared with those corresponding variable domains ofthe human sequences.

The comparison is made by an automated-computarized method (PC-DOS HIBIOPROSIS 06-00, Hitachi.). The most homologous human variable regions arethen compared residue for residue to the corresponding murine regions.This will also define the human subgroup to which each mouse sequencemost closely resambles.

Prediction of T-epitopes.

In the second step, the two homologous variable region sequences, mouseand human are analysed for prediction of T-antigenic sequences.

The algorithm AMPHI (Bersofsky et al., (1987) J. Immunol 138: 2213)predicts α Helical sequences. The algorithm SOHHA predicts the strip ofhelix hydrophobicity (Elliott et al., (1987) J.Immunol. 138: 2949).These algorithms predict T-cell presented fragments of antigenicproteins.

Analysis for immunogenicity reduction.

Those residues in the mouse framework which differ from its humancounterpart are replaced by the residues present in the humancounterpart. This switching occurrs only with those residues which areat the T-antigenic sequences.

Finally, replacement of those residues responsable for the canonicalstructures or those involved in the Vernier zone could have asignificant effect on the tertiary structure. Hence, they can not beincluded in the replacement. Additional information about the influenceof the proposed replacements on tertiary structure or the binding site,could be obtained from a molecular model of the variable regions.

The method for constructing and expressing the altered antibody.

The following procedures are used to prepare recombinant DNA sequenceswhich incorporate the CDRs of the murine mAb, both light and heavychains, into human, frameworks that can be used to transfect mammaliancells for the expression of recombinant antibody less immunogenic andwith the antigen specificity of the animal monoclonal antibody:

a) Mutagenesis and assembly of variable region domains including CDRsand FRs regions. The PCR-mutagenesis method (Kamman et al., (1989)Nucleic Acids Res.17: 5404) is preferably used to introduce the changesat different positions.

b) Preparation of an expression vector including one variable region andthe corresponding human constant region which upon transfection intocells results in the secretion of protein sufficient for affinity andspecificity determinations.

c) Co-transfection of heavy and light chain expression vectors inappropriate cell lines.

After about 2 weeks, the cell supernatants are analyzed by ELISA forhuman IgG production. The samples are then analysed by any method forhuman IgG capable for binding to specific antigens.

Formulation for Performing In Vitro and In Vivo Diagnostic Studies.

The anti CD6 mAbs can be used for in vitro and in vivo diagnosticpurposes in the different clinical forms of Psoriasis, for monitoringpatients after topical or systemic treatments, as well as for predictingrelapses.

These mAbs purified and dissolved in buffer solution (pH 7.0+/−0.5)containing Sodium Azide (0.01-0.2%) and Bovine Serum Albumin (0.05-0.2%)can be used to quantify the CD6+ T lymphocytes and/or the expression ofthis molecule on the surface of the lymphoid cells in biological fluids(i.e. blood, cephaloraquidium liquid, synovial liquid) incubating 50 to200 mL of the sample with 10 to 30 mL of the mAb at concentrationsbetween 0.1 and 3 mg/mL, for 20 to 30 minutes at 4° C. Followed bywashing with buffer solution and incubation with immunoglobulins ofanother animal species conjugated with fluorescent substances (i.e.Fluorescein, Phycoerythrin). The anti CD6 mAbs can be conjugateddirectly with fluorescent substances by different methods (Coligan, J.E. et al. (Ed.) Current Protocols in immunology, National Institutes ofHealth. Vol. I:5.3.2. Wiley Interscience) and be used with similarpurposes previously described, at concentrations between 5 and 30 mg/mL.

Immunohistochemical Evaluation of the Lesions of Patients withPsoriasis.

The immunohistochemical study of cutaneous lesions or other affectedtissues (i.e. articulations) of patients with Psoriasis vulgar can beperformed on tissue cryostat sections (i.e. skin) fixed or not in coldacetone, incubating the anti CD6 mAb dissolved between 3 to 10 mg/mL inbuffer solution (pH7.0+/−0.5) containing Sodium Azide (0.05-0.2%) andBovine Serum Albumin (0.05-0.2%) during 30 minutes. Followed byincubation of the tissue sections with biotinylated anti mouseimmunoglobulins (i.e. from sheep, DAKO) and Avidin Biotin PeroxidaseComplex (i.e. DAKO) during 30 minutes at room temperature. Finallyreaction is developed using 3-amino-9-ethyl-carbazole as chromogen(sigma) (Hsu, S. M. at al. (1981) J. Histochem. Cytochem. 29:577).Biopsies must be examined by two specialists and the evaluation of theCD6 is adjusted to a scale of points <10% (+/−), 10-25% (+), 25-50%(++), 50-90% (+++), 90-100% (++++).

Different antibodies to the T lymphocytes CD can be used, anti CD3 (iort3), anti CD4 (ior t4), anti CD8 (ior t8) and an anti CD45 (ior L3),also an anti Epidermal Growth Factor Receptor mAb (ior egf/r3) as amarker of keratinocytes activation (mozzanica, N. et al. (1994) ActaDerm. Venereol. Suppl. Stockh. 186:171), which allows the evaluation ofdetails of the characteristics of the inflammatory infiltrate of thelesions during the course of the treatment of the disease.

Immunohistochemical Monitoring of the Lesions of the Treated Patients.

Patients treated with anti CD6 monoclonal antibodies can have biopsiesof the lesions performed previous to initiating the treatment, duringthe course of the treatment and after ending the treatment always in thearea next to the initial biopsy to evaluate the therapeutical efficacyof the compositions of topical or systemic use. The scale forclassifying treatment response can be qualitative in per cent and isestablished by the index of CD6+ cells over the total CD45+ cells,evaluated in each biopsy.${{CD6}\text{+}\quad {cells}\quad {index}} = \frac{{Number}\quad {of}\quad {CD6}\text{+}\quad {cells} \times 100}{{Number}\quad {of}\quad {CD45}\text{+}\quad {cells}}$

The index of CD3+ cells, CD4+ cells and CD8+ cells can also beestablished over the total of CD45+ cells and the index of CD4+ and CD8+over the total of CD6+ cells.${{CD3}\text{+}\quad {cells}\quad {index}} = \frac{{Number}\quad {of}\quad {CD3}\text{+}\quad {cells} \times 100}{{Number}\quad {of}\quad {CD45}\text{+}\quad {cells}}$${{CD4}\text{+}\quad {cells}\quad {index}} = \frac{{Number}\quad {of}\quad {CD4}\text{+}\quad {cells} \times 100}{{Number}\quad {of}\quad {CD45}\text{+}\quad {cells}}$${{CD8}\text{+}\quad {cells}\quad {index}} = \frac{{Number}\quad {of}\quad {CD8}\text{+}\quad {cells} \times 100}{{Number}\quad {of}\quad {CD45}\text{+}\quad {cells}}$${{CD4}\text{+}\quad {cells}\quad {index}} = \frac{{Number}\quad {of}\quad {CD4}\text{+}\quad {cells} \times 100}{{Number}\quad {of}\quad {CD6}\text{+}\quad {cells}}$${{CD8}\text{+}\quad {cells}\quad {index}} = \frac{{Number}\quad {of}\quad {CD8}\text{+}\quad {cells} \times 100}{{Number}\quad {of}\quad {CD6}\text{+}\quad {cells}}$

Immunoscintigraphic Monitoring of Treated Patients.

Another form of evaluating the effect of the treatment with the anti CD6mAb can be the immunoscintigraphic study in the patient of theexpression and distribution of the CD6+ calls during the course oftreatment using between 1 and 5 mg of the same mAb conjugated withradioactive isotopes such as 99 m Technetium, using a conjugating methodlike the one described by Mathers, S. J. at al. (1990) J. Nucl. Med.31(5):692.

Obtainment of Therapeutic Formulations for Topical and Systemic Use.

The anti CD6 mAbs can be used with therapeutical purposes in differentclinical forms of Psoriasis, both with topical and systemicformulations, in single or multiple doses, with one or various treatmentcycles according to the severity of the disease.

The topical therapeutic formulations with anti CD6 mAb can be composedby semisolid systems in one or two phases, mainly with hydrophilicformulations that allow the incorporation of the mAb dissolved insterile buffer solution (pH7.0+/−0.5) in doses between 0.1 mg and 5 mgper each gram of the product. Formulations can be elaborated as gels,jelly, ointment, lotions and creams with a liquid matrix (i.e. water)formulated with gelatin, carboximethylcellulose or similar substancesand bases containing glycerine, calcium; additionally the compositionsmay contain preservatives (i.e. p-hydroxibenzoate) to avoidcontamination. The pH must be physiological so as not to affect thecharacteristics of the mAb. These therapeutic compositions should permitthe releasement and penetrability of the mAb in the skin.

Topical treatment should be applied between one and three times a day,over the lesions covered or not and it can be combined with the systemicuse (mainly endoveneous) of the same mAb with doses between 0.1 and 1mg/Kg of patient body weight. It should be diluted in physiologicalsolution for endoveneous use and administered slowly. The endoveneoustreatment can be applied independently of the topical administration.

Clinical Follow Up of Treated Patients.

The clinical evolution of the lesions may be used as the main criteriaof the evaluation of therapeutic efficacy.

The main variables of response used for measuring the effects of thetreatment may be the improvement of the clinical characteristics of thelesions (infiltration, scales, erythema) and the reduction of the areaof the lesions.

The degree of severity of the signs of the disease (infiltration, scalesand erithema) can be established between the values 0-1-2.

0- no sign.

1- scarce presence.

2- intense presence.

The extension of the treated scales should also be considered, measuring2 of its diameters and calculating the area of the lesion by multiplyingthe product of the radios (in centimeters) by p (3.14). The dimension ofthe scale at time 0 represents 100% and in the subsequent evaluationsthe per cent of the dimension of the scale is establishedproportionally.

A PSORIASIS SEVERITY SCORE (PSS) similar to PASI (psoriasis area andseverity index) (Fredriksson, T. et al. (1978) Dermatologica 157:238) isobtained with the formula:$\frac{{{infiltration}{\quad \quad}( {0{–2}} )} + {{scales}\quad ( {0{–2}} )} + {{erithema}\quad ( {0{–2}} )}}{6} \times \% \quad {of}\quad {the}\quad {affected}\quad {area}$

Responsible to treatment is stratified according to the changes in thePSS when completing the times of evaluations, establishing the followingcategories (Perkins, W. et al. (1993) Br. J. Dermatol. 129:584)

Clear (>90% improvement in PSS)

Responders (>50% improvement in PUS)

Non-Responders (<50 1 improvement or deterioration in PSS)

Worsening (>50% increase in PSS)

The evaluation times of the response can be assumed up to 12 weeks fromthe date of initiating the application of the treatment (pretreatment,weeks 1, 2, 3, 4, 6, 8, 12).

EXAMPLE 1 Murine Variable Region of ior t1A Monoclonal Antibodies DNASequencing

Cytoplasmic RNA was extracted from about 10⁶ T1 hybridoma cells asdescribed by Faloro et al (Faloro, J. et al. (1989) Methods inEnzimology 65:718).

The cDNA synthesis reaction consisted of 5 ug RNA, 50 mM Tris-HCl, pH7.5, 75 mM KCl, 10 mM DTT, 3 mM MgCl₂, 25 pmol CG2AFOR primer

(5′GGAAGCTTAGACCGATGGGGCCTGTTGTTTTG 3′) for heavy chain variable regionor CK2FOR (5′GGAAGCTTGAAGATGGATACAGTTGGTGCAGC 3′) for light chainvariable region, 250 uM each of dATF, dTTP, dCTP, dGTP, 15 uribonuclease inhibitor (RNA guard, Pharmacia) in a total volume of 50ul.

Samples were heated at 70° C., for 10 min and slowly cooled to 37° C.over a period of 30 min. Then, 100 units MMLV reverse transcriptase(BRL) were added and the incubation at 37° C. continued for 1 hour.

The VH and VK cDNAs were amplified using the PCR as described by Orlandiet al (Orlandi, R. et al. Proc. Natl. Acad. Sci. USA 86.3333-3837,(1989)). For PCR amplification of VH, DNA/primer mixtures consisted of 5ul cDNA, 25 pmoles CG2A FOR

(5′GGAAGCTTAGACCGATGGGGCCTGTTGTTTTG3′)

and VH1 BACK primers

(5′AGGT(G/C)(A/C)A(A/G)CTGCAG(G/C)AGTC(A/T)GG 3′).

For PCR amplification of VK, DNA/primers mixtures consisted of 5 ulcDNA, 25 pmoles of CK2 FOR

(5′GGAAGCTTGMAGATGGATACAGTTGGTGCAGC 3′) and

VK10BACK (5′TTGAATTCCAGTGATGTTTTGATGACCCA 3)′ primers.

To these mixtures were added 2.5 mM each of dATP, dCTP, dTTP, and dGTP,5 ul constituents of 10X buffer thermolase and 1 unit of Thermolase(IBI)in a final volume of 50 ul. samples were subjected to 25 thermal cyclesat 94° C., 30 sec; 50° C., 30 sec; 72° C., 1 min; and a last incubationfor 5 min at 72° C. Amplified VH and VK DNA were purified on Prep. AGene purification kit (BioRad).

The purified VH and VK DNA were cloned into M13 vector. Clones weresequenced by the dideoxy method using T7 DNA Pol (Pharmacia). See FIGS.1 and 2.

EXAMPLE 2 Modification of the Variable Domain Sequences of ior t1AMurine Monoclonal Antibody to Humanise the Predicted T-cell AntigenicSequences

The variable region sequences of heavy and light chains of ior t1A wereanlyzed for T-cell antigenic sequences. It was made by using thecomputer algorithm AMPHI, which predict segments of the sequences 11amino acids in length with an amphipatic helix structure, that is haveone side hydrophobic and one side hydrophilic which bind to MHC IImolecules.

Onto the variable domain sequence of the heavy chain were predicted 3segments which are: (It is used Kabat's numbering.).

1. FR 1 between amino acids 2-21,

2. CDR1 and FR2 between amino acids 29-43.

3. CDR3 and FR4 between amino acids 95-111.

The FIG. 1 shows the sequences corresponding to heavy chain.

This murine sequence is compared with the immunoglobulin sequencesincluded in the GeneBank and EMBL database. The most homologous humanvariable region sequence is determined and also the human subgroup towhich the murine sequence most closely resembles is defined. In thiscase the human sequence founded was an IgM belonging to subgroup III ofKabat.

Both variable region sequences, human and murine are then comparedresidue for residue and are selected those residues at FR regions whichare not involved in the Vernier zone or with the canonical structures.Therefore they could be changed by those residues at the same positiononto the human sequence. The positions 13 and 19 are not modified due tothe amino acid Lys is present in the same position in other humanimmunoglobulins belonging to the same subgroup.

For the heavy chain of murine ior t1A were proposed 4 replacements:

1. THR at position 40 by ALA.

2. GLU at position 42 by GLY.

3. THR at position 108 by LEU

4. LEU at position 109 by VAL.

The game procedure applied to the light chain (FIG. 2) rendered a set ofoverlaping segments from aminoacid 2 aminoacid 69.

After the analysis we proposed 7 replacements in FRs 1 and 2 atpositions: 3, 11, 12, 15, 17, 41 and 43.

1. LYS at position 3 by GLN

2. MET at position 11 by LEU

3. TYR at position 12 by SER

4. LEU at position 15 by VAL

5. GLU at position 17 by ASP

6. TRP at position 41 by GLY

7. SER at position 43 by ALA

EXAMPLE 3 Construction of Mutant Heavy and Light Chain Variable Regionof ior t1A by PCR Mutagenesis

The changes in the amino acids of mutant heavy and light chain variableregion were constructed using PCR mutagenesis (Kammann, M. et al. (1989)Proc. Natl. Acad. Sci. USA, 86: 4220).

Briefly: Two amplification by PCR: the reaction mixture was: 0.5 ul theVH supernatant of single strand DNA cloned in M13, 25 pmoles mutagenicoligo 1 or 2, 25 pmoles mutagenic oligo 3 or 4 primers (See below theprimers sequences). To these mixtures were added 2.5 mM each of dATF,Dttp, dCTP, and dGTP, 5 μl constituents of 10X Vent Polymerase buffer(NSB) and 1 unit of Vent DNA Polymerase (NEB) in a final volume of 50ul. Samples were subjected to 12-15 thermal cycles at 94° C., 30 sec;50° C., 30 sec; 75° C., 1 min; and a last incubation for 5 min at 75° C.The products of both PCRs are joined in a second PCR using the outsideprimers only (3 and 4). Amplified VH DNA were purified on Prep. A Genepurification kit (BioRad).

For the changes in the heavy chain, FR1 in the positions 5, 7, 11, 12and 13 the primers used, were:

Primer 1:

5′TGG GTT CGC CAG GCT CCG GGG AAG AGG CTG GAG 3′.

Primec 3;

5′GTA AMA CGA CGG CCA GT 3′.

These primers are combined in one PCR.

Primer 2:

5′CTC CAG CCT CTT CCC CGG AGC CTG GCG AAC CCA 3′.

Primer 4:

5′AGC GGA TAA CAA TTT CAC ACA GGA 3′.

These primers are combine in one PCR. Then, the products of both PCRsare combined in one PCR using 3 and 4 primers.

For the changes in the POSITION 108 and 109, the primers designed were:

Primer 1:

5′GGC CAA GGC ACC CTT GTC ACC GTC TCC 3′.

Primer 3:

5′GTA AAA CGA CGG CCA GT 3′.

These primers are combined in one PCR.

Primer 2:

5′GGA GAC GGT GAC AAG GGT GCC TTOG GCC 3′.

Primer 4:

5′AGC GGA TAA CAA TTT CAC ACA GGA 3′.

These primers are combined in one PCR. Then, the products of both PCRsare combined in one PCR using 3 and 4 primers.

In the light chain for the changes in the FR1 in the residues 3, 11, 12,15 and 17, the primers were designed as:

PRIMER 1;

5′TGT GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCG CAT CGGTGG GAG ACA GAG TCA CC 3′

PRIMER 3: 5′GTA AAA CGA CGG CCA GT 3′

These primers are combined in one PCR

PRIMER 2:

5′GGT GAC TCT GTC TCC CAC CGA TGC AGA CAG GGA GGA TGG AGA CTG GGT CATCTG GAT GTC ACA 3′

PRIMER 4: 5′ACT GGC CGT CGT TTT TAC 3′

These primers are combined in one PCR.

The products of both PCRs are combined in one PCR using primers 3 and 4.

For the changes in the residues 41 and 43 in the FP2 the primers were:

PRIMER 1: 5′CAG AAA CCA GGG AAA GCT CCT AAG ACC CTG 3′

PRIMER 3: 5′GTA AAA CGA CGG CCA T 3′

These primers are combined in one PCR.

PRIMER 2: 5′CAG GGT CTT AGG AGC TTT CCC TGG TTT CTG 3′

PRIMER 4: 5′ACT GGC CGT CGT TTT TAC 3′

These primers are combined in one PCR.

The products of both PCRs are combined in one using primers 3 and 4.

EXAMPLE 4 Pharmaceutical Formulation for Topic Use in Cutaneous Lesionsof Patients with Psoriasis Vulgar

The ior t1 mAb was purified and dissolved (50.00 mg) in sterile buffersolution (10 mL pH7.0+/−0.5) containing Monobasic Sodium Phosphate 4.50mg , Dibasic Sodium Phosphate 18.00 mg , Sodium Chloride 86.00 mg,Polysorbate 80 1.952 μL, Water for injection. The solution containingthe mAb was added to a jelly base.

The therapeutic jelly was elaborated with a buffer solution having acomposition similar to the one described for the mAb (pH 7.0+/−0.5).

EXAMPLE 5 Histological Study of Cutaneous Lesions of Patients withPsoriasis Vulgar

The immunohistochemical study of the cutaneous lesions of patients withPsoriasis vulgar was performed on cryostat sections of skin tissue,using the mAb ior t1 in parallel with different mAbs directed against CDof T lymphocytes. mAbs ior t3 (anti CD3), ior t4 (anti CD4), ior t8(anti CD8), ior L3 (anti CD45) and DAKO CD6 (anti CD6), as control, aswell as ior egf/r3 (anti Epidermal Growth Factor Receptor) were used.Followed by incubation of tissue sections with biotinylated anti mouseimmunoglobulins (i.e. from sheep, DAKO) and Avidin Biotin PeroxidaseComplex (i.e. DAKO). Finally reaction is developed using3-amino-9-ethyl-carbazole as chromogen (Sigma). Biopsies were examinedby two specialists and the evaluation of the CD6 was adjusted to a scaleof points <10% (+/−), 10-25% (+), 25-50% (++), 50-90% (+++), 90-100%(++++).

Patients treated with anti CD6 monoclonal antibodies had biopsies of thelesions performed previous to initiating the treatment and at the end ofthe third week of treatment in the area next to the initial biopsy . Thescale for classifying treatment response was qualitative in per cent andestablished by the index of CD6+ cells over the total CD45+ cells ,evaluated in each biopsy. The per cent of the expression of the antiEpidermal Growth Factor receptor (EGF-R) in the different stratus of theskin was determined as well as the modifications of the histologicalcharacteristics typical of the disease.

In the inflammatory infiltrate characteristic of Psoriasis Vulgar wasfound to exists an important Expression (+++) of the CD6+ T lymphoidphenotype.

The CD3+ lymphocytes represent approximately 100% of the CD45+ cells.

The CD6+ cells represents approximately between 60% and over 90% of theCD3+ cells.

The CD6+ cells (ior t1) represents approximately between 30% and 50% ofthe CD6+ cells (DAKO CD6).

The expression of the EGF-R in keratinocytes was elevated showing areticulated pattern.

The predominance of the expression of the CD6 molecule in the Tlymphocytes of the inflammatory infiltrate results significant since itis a molecule characteristic of activated T lymphocytes. This makes usbelieve that this might constitute a leukocitary adhesion molecule ofthe T lymphocytes, initially activated in the skin by the penetration ofexogenous antigens or modified self antigens. Necessary this for theinteraction with specific cellular determinants of the skin activatedduring the response to the said antigens.

EXAMPLE 6 Clinical Response of Patients with Psoriasis Vulgar to theTopical Therapy with ior t1 mAb

A clinical trial of patients with a diagnosis of Psoriasis Vulgar inrelapse with lesions characteristic of this disease was performed. Thestudy was scheduled in two groups of fourteen patients per group difinedaccording to the jelly received for topical treatment (ior t1 mAb orvehicle). The topical therapeutic formulation was conformed by a jellybase or vehicle (Sodium Carboximethylcelulose v/v Propilenglycol,Methylparabeno, Trietanolamine) in which the mAb was incorporated.Treatment was applied two times a day during 21 days without occlusionof the lesions treated.

Psoriatic plaque lesions whiten in all the patients treated with ior t1mAb (FIG. 4). This result corresponds with post treatment biopsyperformed 21 days after initiating application of the mAb. A decrease ofT lymphocyte infiltrate and of the expression of the EGF-R inkeratinocytes as well as a regression in the histological signscharacteristic of the disease were observed.

EXAMPLE 7 Clinical Response of Psoriasis Vulgaris Patients AfterScaling-down Topical Treatment with ior t1 Monoclonal Antibody

A Pilot Clinical Trial in 19 confirmed long-lasting psoriasis vulgarispatients with more than 10% and less than 25% of their skin affected wasperformed. Three different groups of 6, 7 and 6 patients received atherapeutic topical formulation containing 0.3, 1 and 3 mg of ior t1AmAb/gram of jelly respectively, in a vehicle jelly consisting of SodiumCarboximethylcelulose A/V, Propylenglicol, Methylparabene,Propylparabene and Triethanolamine. Patients were topicaly treated 2times a day during 21 days.

PASI (Psoriatic Area and Severity Index) was scored and analised andhuman anti mouse antibody (HAMA) response in the sera of patients wasalso studied. The best results related to clinical response (PASI) anddisease free interval were obtained in the group treated with the loweramount of ior t1 mAb (0.3 mg), as well as the HAMA (Human Anti-MouseAntibody) titres and the amount of patients by group developing it werealso higher in that group. Moreover, the presence of anti-idiotypeantibodies in patient's sera studied by means of blocking ELISA (EnzymeLinked Immunosorbent Assay) and FACS (Fluorescent Activating CellSorter) were more frequent and much higher in those patients treatedwith the lower doses of 0.3 mg per gram of jelly.

EXAMPLE 8 Clinical Response of one Patient with the Severe Form ofGeneralized Psoriasis to the Endoveneous Treatment with the ior t1 mAb.

Female patient, 56 years old with a history of Psoriasis with psoriaticarthropathy diagnosed approximately 17 years ago. In the last 5 yearsthe patient has suffered frequent and intense crisis, causing frequenthospitalizations. Crisis started with erithematosquamous generalizedlesions, pain in articulations and muscles, feverish, generalized edemasand malaise. This general status persists and 21 days later new lesionsappear in the axilary region, the neck and around the breasts, withulcerations and infected serose secretion accompanied by fever. Due tothe torpid evolution of the disease and the intolerance to all previoustreatments including steroid creams, treatment with methotrexate isindicated administering 3 cycles with a total doses of 15 mg. Noclinical response was observed.

Single endoveneous dose of ior t1 mAb at 0.6 mg/Kg of body weight,administered slowly, diluted in 200 mL of Saline Solution 0.9.

Simultaneously a therapeutical jelly containing ior t1 mAb at aconcentration of 3 mg of mAb/ g of jelly was applied 2 Times a day inall the lesions during two days.

The patient starts to improve her general status and dermatologicalpicture around the 6th day of treatment. At day 21 an evaluation wasperformed and 60% of the body surface was without lesions and the restof the skin showed improvement of the clinical signs of the disease. Thepatient referred improvement of the symptoms in articulations, having agood general status, normal vital signs and routine laboratory tests.

After 30 days the patient maintains complete regression of the symptomsof the disease.

A viable culture of T1 hybridoma cells, as described in theabove-identified application, namely IOR-T1A, was deposited inaccordance with the Budapest Treaty with the European Collection of CellCultures, Centre for Applied Microbiology and Research, Salisbury,Wiltshire, SP4 OJG, United Kingdom, on Nov. 26, 1996. The accessionnumber is for this deposit is 96112640. All restrictions and conditionsby the depositors/applicants upon availability of the cell culture tothe public will be irrevocably removed upon granting of a patent basedon the above-identified application.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 and 2

Analysis for the modification by way of humanization of the variableregions of heavy and light chains of monoclonal antibody ior t1A:

FIG. 1: Sequence of the variable region of heavy chain of the murine iort1A monoclonal antibody.

FIG. 2: Sequence of the variable region of light chain of the murine iort1A monoclonal antibody.

A: Sequence of the variable region of heavy chain or light chain of iort1A murine mAb.

B: Sequence of the variable region of the most homologous humanimmunoglobulin.

C: Sequence of the modified variable region of ior t1A.

shading: predicted T-cell antigenic sequences.

Underlined amino acids residues: amino acids involved in tertiarystructure.

Bold font: complementarity determining regions.

Amino acids residues in boxes: replacements which are proposed.

The description is idem for both, heavy and light chains.

FIG. 3

The results of the expression of the CD6 antigens in lymphocytes of theinflammatory infiltrates characteristic of the cutaneous lesions ofpatients with Psoriasis Vulgar are shown. This evaluation was performedin cryostat sections biopsies of skin affected by plaques lesionslocalized in the upper and/or lower limbs and/or thorax-abdomen. Thehistological evaluation was performed previous to theimmunohistochemistry study.

FIG. 4

The evaluation of the therapeutic efficacy of the anti CD6 mAbs used inthe treatment of the Psoriasis was performed considering the followingvariables: infiltration, scales, erythema and the size of the area ofthe lesion. The great of severity was established between the valueszero-1-2. The extension of the treated plaques was established measuringto of its diameters. A Psoriasis Severity Score (PSS) similar to thePASI (Psoriasis Area and Severity Index) was obtained and the responseto treatment was stratified according to the changed in the PSS at theend of the designated evaluation time. The following categorizes wasestablished: Clear, Responders, Non-Responders and Worsening.

FIG. 5

Clinical response of patients treated with a topical formulationcontaining 0.3, 1 or 3 mg of ior t1A mAb/gram of jelly respectively wasevaluated by PASI (Psoriatic Area and Severity Index) and the human antimouse antibody (HAMA) response in the sera of these patients was alsostudied. The best results related to clinical response (PASI) anddisease free interval were obtained in the group treated with the loweramount of ior t1 mAb (0.3 mg), as well as the HAMA titres and the amountof patients by group developing it were also higher in that group.Moreover, the presence of anti-idiotype antibodies in patient's serawere more frequent and much higher in those patients treated with thelower doses of 0.3 mg per gram of jelly.

FIG. 6

Endovenous treatment was applied with the ior t1 mAb to a 56 years oldpatient with a history of Psoriasis with psoriatic arthropathy diagnosedapproximately 17 years ago. Presenting now a severe form of generalizedpsoriasis characterized by erithematosquamous generalized lesions, painin articulations and muscles, feverish, generalized edemas and malaise.This general status did not respond to treatment including methotrexate.Treatment was performed with single endoveneous dose of ior t1 mAb at0.6 mg/Kg of body weight, administered slowly, diluted in 200 mL ofSaline Solution 0.9%. Simultaneously a therapeutical jelly containingior t1 mAb at a concentration of 3 mg of mAb/ g of jelly was applied 2times a day in all the lesions during two days, for a total of 224 g. oftherapeutic jelly. The clinical response and the immunohistochemistrylaboratory results were evaluated weekly. Photographs of the evolutionof the cutaneous lesions are shown (the day before and 21 days after thetreatment).

In accordance with the invention, there are provided monoclonalantibodies recognizing human CD6 in accordance with claims 1 or 2,wherein the subclon ior t1A obtained from the hybridoma of the same name(Deposit Number: pending) has a variable region of its heavy chain ofsequence:

GLU VAL GLN LEU VAL GLU SER GLY GLY GLY LEU VAL LYS PRO GLY GLY SER LEULYS LEU SER CYS ALA ALA SER GLY PHE LYS PHE SER ARG TYR ALA MET SER TRPVAL ARG GLN THR PRO GLU LYS ARG LEU GLU TRP VAL ALA THR ILE SER SER GLYGLY SER TYR ILE TYR TYR PRO ASP SER VAL LYS GLY ARG PHE THR ILE SER ARGASP THR SER SER ASN THR ALA TYR MET GLN LEU SER SER LEU ARG SER GLU ASPTHR ALA MET TYR TYR CYS ALA ARG ARG ASP TYR ASP LEU ASP TYR PHE ASP SERTRP GLY GLN GLY THR THR LEU THR VAL SER SER and the variable region ofits light chain of sequence: ASP ILE LYS MET THR GLN SER PRO SER SER METTYR ALA SER LEU GLY GLU ARG VAL THR ILE THR CYS LYS ALA SER ARG ASP ILEARG SER TYR LEU THR TRP TYR GLN GLN LYS PRO TRP LYS SER PRO LYS THR LEUILE TYR TYR ALA THR SER LEU ALA ASP GLY VAL PRO SER ARG PHE SER GLY SERGLY SER GLY GLN ASP TYR SER LEU THR ILE SER SER LEU GLU SER ASP ASP THRALA THR TYR TYR CYS LEU GLN HIS GLY GLU SER PRO PHE THR PHE GLY SER GLYTHR LYS LEU GLU ILE LYS ARG ALA

In accordance with the invention, there are also provided monoclonalantibodies recognizing human CD6 in accordance with claims 1 to 3, whichis a humanized variant of subclon ior t1A, and a variable region of itsheavy chain has the sequence:

GLU VAL GLN LEU VAL GLU SER GLY GLY GLY LEU VAL LYS PRO GLY GLY SER LEULYS LEU SER CYS ALA ALA SER GLY PHE LYS PHE SER ARG TYR ALA MET SER TRPVAL ARG GLN ALA PRO GLY LYS ARG LEU GLU TRP VAL ALA THR ILE SER SER GLYGLY SER TYR ILE TYR TYR PRO ASP SER VAL LYS GLY ARG PHE THR ILE SER ARGASP ASN VAL LYS ASN THR LEU TYR LEU GLN MET SER SER LEU ARG SER GLU ASPTHR ALA MET TYR TYR CYS ALA ARG ARG ASP TYR ASP LEU ASP TYR PHE ASP SERTRP GLY GLN GLY THR LEU VAL THR VAL SER SER and the variable region ofits light chain has the sequence: ASP ILE GLN MET THR GLN SER PRO SERSER LEU SER ALA SER VAL GLY ASP ARG VAL THR ILE THR CYS LYS ALA SER ARGASP ILE ARG SER TYR LEU THR TRP TYR GLN GLN LYS PRO GLY LYS ALA PRO LYSTHR LEU ILE TYR TYR ALA THR SER LEU ALA ASP GLY VAL PRO SER ARG PHE SERGLY SER GLY SER GLY GLN ASP TYR SER LEU THR ILE SER SER LEU GLU SER ASPASP THR ALA THR TYR TYR CYS LEU GLN HIS GLY GLU SER PRO PHE THR PHE GLYSER GLY THR LYS LEU GLU ILE LYS ARG ALA

In addition, the Information for SEQ ID: 1 and the Sequence Descriptionfor SEQ ID NO:1, as well as the same Information and SequenceDescription for ID Nos. 2-4 are all incorporated herein by reference andis exactly as set forth in the computer readable form (CRF) disc of the“Sequence Listing” or in the substitute paper copy of the “SequenceListing”.

“As referenced hereinabove in the descriptions of FIGS. 1-2 of theinstant patent application, the sequences in lines or rows B correspondto human sequences of the variable regions with a high homology with themurine anti-CD6 antibody. These sequences are conventional andwell-known in the art, and in the present patent application they wereonly used therein so as to be compared with the sequences of the murineantibody in the procedure to humanize the murine anti-CD6. Thus, thesehuman sequence are not claimed nor do they form any part of the presentinvention as defined and claimed in the case.

In the present application, applicants claim, as their invention, thesequences for heavy and light chains of the murine antibody (sequences 1and 2), and the humanized version of said antibody (sequences 3 and 4).More particularly, these sequences are:

Sequence 1: represents the murine variable region of the heavy chain(anti-CD6 antibody);

Sequence 2: represents the murine variable region of the light chain(anti-CD6 antibody);

Sequence 3: represents the humanized variable region of the heavy chain(anti-CD6 antibody); and

Sequence 4: represents the humanized variable region of the light chain(anti-CD6 antibody).

All four sequences of the present invention are unique sequences whichare all novel and which are believed to be patentable.”

4 119 Amino acid residues. Amino acid. Unknown. Unknown. Protein No No-N Terminal fragment. Mice Balb/C ior t1A Murine hibridoma Sub-clone iort1A Experimental. Sequence corresponding to the variable region of theheavy chain of the monoclonal antibody recognizing human CD6, designatedas sub-clone ior t1A. 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu ValLys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly PheLys Phe Ser Arg Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Thr Pro Glu LysArg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Tyr Ile TyrTyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn ValLys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu AspThr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Asp Leu Asp Tyr PheAsp Ser Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 109Amino acid residues. Amino acid. Unknown. Unknown. Protein Yes No -NTerminal fragment. Mice Balb/C ior t1A Murine hibridoma Sub-clone iort1A Experimental. Sequence corresponding to the variable region of thelight chain of the monoclonal antibody recognizing human CD6, designatedas sub-clone ior t1A. 2 Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met TyrAla Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser ArgAsp Ile Arg Ser Tyr 20 25 30 Leu Thr Trp Tyr Gln Gln Lys Pro Trp Lys SerPro Lys Thr Leu Ile 35 40 45 Tyr Tyr Ala Thr Ser Leu Ala Asp Gly Val ProSer Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr IleSer Ser Leu Glu Ser 65 70 75 80 Asp Asp Thr Ala Thr Tyr Tyr Cys Leu GlnHis Gly Glu Ser Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu IleLys Arg Ala 100 105 119 Amino acid residues. Amino acid. Unknown.Unknown. Protein No No -N Terminal fragment. Animal cells. NSO “ SP 2/0” CHO Sub-clone ior t1A By similarity with known sequence. Sequencecorresponding to the humanized variant of sub-clone ior t1A recognizinghuman CD6, particularly to the variable region of its heavy chain. 3 GluVal Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr 65 70 7580 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 9095 Ala Arg Arg Asp Tyr Asp Leu Asp Tyr Phe Asp Ser Trp Gly Gln Gly 100105 110 Thr Leu Val Thr Val Ser Ser 115 109 Amino acid residues. Aminoacid. Unknown. Unknown. Protein No No -N Terminal fragment. Animalcells. NSO “ SP 2/0 ” CHO Sub-clone ior t1A By similarity with knownsequence. Sequence corresponding to the humanized variant of sub-cloneior t1A recognizing human CD6, particularly to the variable region ofits light chain. 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser AlaSer Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg AspIle Arg Ser Tyr 20 25 30 Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala ProLys Thr Leu Ile 35 40 45 Tyr Tyr Ala Thr Ser Leu Ala Asp Gly Val Pro SerArg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile SerSer Leu Glu Ser 65 70 75 80 Asp Asp Thr Ala Thr Tyr Tyr Cys Leu Gln HisGly Glu Ser Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile LysArg Ala 100 105

What is claimed is:
 1. A monoclonal antibody that is a humanized subclone of IOR-T1A produced by the hybridoma deposited with ECACC, CAMRunder accession number 96112640, having heavy and light chains withhumanized variable regions.
 2. A monoclonal antibody according to claim1, comprising: a heavy chain having a variable region with the followingsequence: Glu Val GIn Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro GlyGly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Arg Tyr AlaMet Ser Trp Val Arg GIn Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr lleSer Ser Gly Gly Ser Tyr lle Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thrlle Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr Leu GIn Met Ser Ser Leu ArgSer Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Asp Tyr Asp Leu Asp TyrPhe Asp Ser Trp Gly GIn Gly Thr Thr Leu Thr Val Ser Ser (SEQ. ID. NO. 1)and a light chain having a variable region with the following sequence:Asp lle Lys Met Thr GIn Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly Glu ArgVal Thr lle Thr Cys Lys Ala Ser Arg Asp lle Arg Ser Tyr Leu Thr Trp TyrGIn GIn Lys Pro Trp Lys Ser Pro Lys Thr Leu lle Tyr Tyr Ala Thr Ser LeuAla Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly GIn Asp Tyr SerLeu Thr lle Ser Ser Leu Glu Ser Asp Asp Thr Ala Thr Tyr Tyr Cys Leu GInHis Gly Glu Ser Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu lle Lys ArgAla (SEQ. ID. NO. 2).
 3. A humanized variant of sub-clone IOR T1A ofIgG1 isotype of the monoclonal antibody according to claim 1,comprising: a heavy chain having a variable region with the followingsequence: Glu Val GIn Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro GlyGly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Arg Tyr AlaMet Ser Trp Val Arg GIn Ala Pro Gly Lys Arg Leu Glu Trp Val Ala Thr lleSer Ser Gly Gly Ser Tyr lle Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thrlle Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr Leu GIn Met Ser Ser Leu ArgSer Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Asp Tyr Asp Leu Asp TyrPhe Asp Ser Trp Gly GIn Gly Thr Leu Val Thr Val Ser Ser (SEQ. ID. NO. 3)and a light chain having a variable region with the following sequence:Asp lle GIn Met Thr GIn Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp ArgVal Thr lle Thr Cys Lys Ala Ser Arg Asp lle Arg Ser Tyr Leu Thr Trp TyrGIn GIn Lys Pro Gly Lys Ala Pro Lys Thr Leu lle Tyr Tyr Ala Thr Ser LeuAla Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly GIn Asp Tyr SerLeu Thr lle Ser Ser Leu Glu Ser Asp Asp Thr Ala Thr Tyr Tyr Cys Leu GInHis Gly Glu Ser Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu lle Lys ArgAla (SEQ. ID. NO. 4).
 4. Monoclonal antibodies according to claim 1,which are IgG2a isotype.
 5. Humanized monoclonal antibodies according toclaim 1 which are IgG1 isotype.
 6. Pharmaceutical composition for thetreatment of Psoriasis which contains the monoclonal antibodiesaccording to any one of claims 2 to
 3. 7. Monoclonal antibodiesaccording to claim 2, which are IgG2a isotype.